CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Abstract

Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.

NOT Gate Genetic Circuits to Control Gene Expression in Cyanobacteria

“…30 Regulation based on CRISPRi in which dCas9 and/or the guide RNAs were controlled by a tetracycline-regulated promoter repressed the expression of a fluorescent reporter protein by up to 94% in Synechocystis PCC 6803 4 and by up to 95% in Synechococcus PCC 7002. 5 In comparison, the results presented here show that expression ratios decreased by up to 87% using the P trc2O /LacI q NOT gates controlled with riboswitch B in Synechocystis PCC 6803 and decreased by up to 98% using the P van /VanR NOT gates in S. elongatus . However, we note that CRISPRi based systems have potential advantages as they can be used to repress multiple genes at once, without the need to engineer multiple individual promoters.…”

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

“…CRISPRi holds great promise for a wide range of applications in microorganisms, including bacterial cell growth control [35], genetic screen [25, 36], synthetic biology module development [37, 38] or metabolic networks control in various microorganisms such as E. coli [24, 39, 40], mycobacteria [41], Bacillus subtilis [42], Corynebacterium glutamicum [43], Clostridium beijerinckii [44], yeast [45] and cyanobacteria [7]. In particular, a number of recent studies have exploited CRISPRi to regulate the metabolic pathways in E. coli for enhanced production of various biotechnological products including poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [23], terpenoid [8], pinosylvin [46], flavonoid [47] and mevalonate [48].…”

“…Furthermore, many cyanobacteria possess multiple copies of chromosomes and foreign genes need to be integrated and selected, hence making it much more labor-intensive and time-consuming to establish the CRISPRi system in cyanobacteria. To our best knowledge, only 2 very recent studies have employed CRISPRi to engineer cyanobacteria [7, 27]. Yao et al explored CRISPRi in Synechcocystis sp.…”

“…Sometimes deletion of certain genes essential for metabolic balances is not feasible or easily achieved as the deletion might be lethal to the cells. Furthermore, in many cases intermediate levels of enzyme expression may result in better product titer [7]. Therefore, tunable and balanced gene expression is desirable for high productivity, product titer, and conversion yield, and controllable gene repression/knockdown may be preferable than gene deletion for certain biotechnological applications and synthetic biological manipulations [8].…”

“…PCC 6803 [27] and Synechococcus sp. PCC 7002 [7]. However, whether CRISPRi functions in PCC 7942 has yet to be explored.…”